Title
Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry
Author
Carol, J.
Gorseling, M.C.J.K.
de Jong, C.F.
Lingeman, H.
Kientz, C.E.
van Baar, B.L.M.
Irth, H.
TNO Defensie en Veiligheid
Publication year
2005
Abstract
A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%. © 2005 Elsevier Inc. All rights reserved.
Subject
Biological toxins
Liquid chromatography
Mass spectrometry
On-line digestion
Size-exclusion chromatography
Bovine serum albumin
Cholera toxin
Myoglobin
Staphylococcus enterotoxin
Article
Desalination
Digestion
Electrospray mass spectrometry
Gel permeation chromatography
Nonhuman
Priority journal
Protein analysis
Protein determination
Reversed phase high performance liquid chromatography
Amino Acid Sequence
Cholera Toxin
Chromatography, Gel
Chromatography, Liquid
Enterotoxins
Molecular Sequence Data
Protein Denaturation
Proteins
Spectrometry, Mass, Electrospray Ionization
Bovinae
Chemicals / CAS: Cholera Toxin, 9012-63-9; enterotoxin B, staphylococcal, 39424-53-8; Enterotoxins; Proteins
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DOI
https://doi.org/10.1016/j.ab.2005.08.023
TNO identifier
238786
ISSN
0003-2697
Source
Analytical Biochemistry, 346 (1), 150-157
Document type
article